1. Field of the Invention
This invention relates to a nucleic acid amplifier. More particularly, the invention relates to a nucleic acid amplifier and the like used for gene analyses such as gene expression analysis, screening for infectious diseases, SNP analysis and the like.
2. Description of the Related Art
In recent years, hybridization detection techniques including DNA chips or DNA microarrays have been advanced in practice. DNA chips are ones wherein a wide variety of DNA probes are accumulated and fixed on a substrate surface. When this DNA chip is used for detection of hybridization on the DNA chip substrate surface, gene expression in cells, tissues or the like can be comprehensively analyzed.
Data obtained from the microarray is verified by carrying out a nucleic acid amplification reaction such as a PCR (polymerase chain reaction) process or the like. This is a standard technique of qualitative analysis of a trace amount of nucleic acid. Although techniques other than the PCR process have been used as a nucleic acid amplification technique used for the qualitative analysis of a trace amount of nucleic acid, a real time PCR process is illustrated herein by way of an example.
The real time PCR process is a process wherein DNA or the like is amplified to several hundreds of thousands times by continuously conducting an amplification cycle of “thermal denaturationannealing with a primerpolymerase elongation reaction”. The PCR amplified product obtained in this way is monitored in real time to qualitatively analyze such a trace amount of nucleic acid as mentioned above. In this real time PCR process, a dedicated device integrated of a thermal cycler and a spectrophotofluorometer is used to monitor the PCR amplified product in real time.
The detection method of this real time PCR is described below.
Initially, there is mentioned an intercalator method using SYBR (registered trade name) Green I. In the intercalator method, an intercalator having such a property as to emit fluorescence by binding with double-stranded DNA. This intercalator is bound to double-stranded DNA formed during the course of the PCR reaction, against which excitation light is irradiated thereby emitting fluorescence. The detection of this fluorescence intensity enables an amount of the PCR amplified product to be monitored. According to this intercalator method, there is no necessity of designing and synthesizing a fluorescence-labeled probe specific to a target and thus, this method can be simply utilized for the measurement of a variety of targets.
If it is desirable that arrays whose structures are similar to one another be distinctly detected or if multiplex detection is necessary such as for typing of SNPs, a probe method is used. For the probe method, mention is made, for example, of a TaqMan (registered trade name) probe method wherein an oligonucleotide with its 5′ terminal modified with a fluorescent substance and 3′ terminal modified with a quencher substance is used as probe II.
The TaqMan probe is hybridized specifically to template DNA in an annealing step, under which when excitation light is irradiated, the light is quenched owing to the existence of the quencher substance on the probe, resulting in no emission of fluorescence. However, in the elongation reaction step, the TaqMan probe hybridized to the template DNA is decomposed by the 5′3′ exonuclease activity of the TaqMan polymerase. Eventually, the fluorescent substance is released from the probe, so that the inhibition with the quencher is removed thereby emitting fluorescence. The amount of the PCR amplified product can be monitored by detecting the intensity of the fluorescence.
The procedure of quantitatively determining a gene expression level by the above method using real time PCR is described in more detail. First, PCR is carried out using, as a template, serially diluted standard samples whose concentrations are known to obtain the number of threshold cycles (Ct value) arriving at a given amount of an amplified product. This Ct value is taken as an abscissa and an initial amount of DNA is plotted as an ordinate, thereby providing a calibration curve. Based on this, a sample whose concentration is unknown is subjected to the PCR reaction under similar conditions to obtain a Ct value. An amount of target DNA in the sample is determined from the Ct value and the calibration curve.
Technologies concerning a temperature control and the like at the time of the amplification reaction are disclosed, as techniques concerning the above methods, in JP-A-2003-525617 and Japanese Patent Laid-open No. 2001-136954.